Summary Biological systems are inherently complex and interrelated, as they organize and function through a series of hierarchical networks involving multiple interacting components. Hence, simultaneously visualizing a large number of distinct molecular species inside living cells has become indispensable for understanding these biological processes in a holistic manner. As we enter the era of systems biology, such super-multiplex imaging capability will be transformative across various fields including revealing structure?function relationships in nervous systems; understanding tumor heterogeneity; studying macromolecules choreography during cell regulation, as well as revealing intricate interactions among various organelles of living cells. The goal of this project is to develop a general super-multiplex optical microscopy platform for simultaneously imaging a large number (more than 20) of specific molecular targets inside live cells, an important but otherwise intractable goal by conventional methods such as fluorescence. To do so, we propose to couple the emerging electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy, offering nanomolar detection sensitivity and narrow chemical specificity, with novel vibrational probes consisting of triple-bond-conjugated light- absorbing dyes. The first-generation technique has been recently published, demonstrating a record of 24-color imaging in biological systems (L. Wei ? W. Min. Nature, 544, 465, 2017). Moving towards the next-generation technology, we have laid out systematic plans as to how to crystallize this concept into a much more powerful platform to achieve high-speed, high- sensitivity, super-multiplex vibrational imaging of specific proteins and organelles in living cells. We propose to construct new microscope instrumentations to significantly boost the imaging speed by orders of magnitude (Specific Aim 1), and engineer novel epr-SRS vibrational probes with expanded color palette, superior detection sensitivity, organelle targeting specificity and genetic encodability to specific proteins (Specific Aim 2). Accompanied by these technical developments, we will then apply it to probe systems-level interactions within multiple organelles and proteins during dynamical processes of cytokinesis and apoptosis (Specific Aim 3). If successfully implemented, we will establish a transformative imaging platform that could allow researchers to interrogate an unprecedented large number of bio-molecules in living cells with superb sensitivity, targeting specificity, labeling versatility, and biocompatibility. The resulting super-multiplex optical microscopy would find wide applications in unraveling complex biological systems such as cell biology, neurobiology, immunology, and tumor biology.